Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNA transcripts are blocked or “capped” at their 5′ terminus. In addition to mRNA, some other forms of eukaryotic RNA, such as but not limited to, small nuclear RNA (“snRNA”) and pre-micro RNA (i.e. “pre-miRNA”, the primary transcripts that are processed to miRNA) are also capped.
A “cap” is a guanine nucleoside that is joined via its 5′-carbon to a triphosphate group that is, in turn, joined to the 5′-carbon of the most 5′-nucleotide of the primary mRNA transcript, and in most eukaryotes, the nitrogen at the 7 position of guanine in the cap nucleotide is methylated. Such a capped transcript can be represented as m7G(5′)ppp(5′)N1(pN)x—OH(3′), or more simply, as m7GpppN1(pN)x, where m7G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5′ carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, and N1(pN)x—OH(3′) represents the primary RNA transcript, of which N1 is the most 5′-nucleotide.
The 5′ caps of eukaryotic cellular and viral mRNAs (and some other forms of RNA) play important roles in RNA stability and processing. For example, the cap plays a pivotal role in mRNA metabolism, and is required to varying degrees for processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein.
The 5′ cap structure is involved in the initiation of protein synthesis of eukaryotic cellular and eukaryotic viral mRNAs and in mRNA processing and stability in vivo (e.g., see, Cell 9: 645-653, 1976; Furuichi, et al., Nature 266: 235, 1977; Federation of Experimental Biologists Society Letter 96: 1-11, 1978; Prog. Nuc. Acid Res. 35: 173-207, 1988). Specific cap binding proteins exist that are components of the machinery required for initiation of translation of an mRNA (e.g., see Cell 40: 223-24, 1985; Prog. Nuc. Acid Res. 35: 173-207, 1988). The cap of mRNA is recognized by the translational initiation factor eIF4E (Gingras, et al., Ann. Rev. Biochem. 68: 913-963, 1999; Rhoads, R E, J. Biol. Chem. 274: 30337-3040, 1999). Thus, RNA prepared (e.g., in vitro) for introduction into eukaryotic cells (e.g., via microinjection into oocytes or transfection into cells) should be capped.
Also, many viral RNAs are infectious only when capped, and uncapped RNAs introduced into cells via transfection or microinjection are rapidly degraded by cellular RNases (e.g., see Krieg, and Melton, Nucleic Acids Res. 12: 7057, 1984; Drummond, et al. Nucleic Acids Res. 13: 7375, 1979).
The 5′ cap structure provides resistance to 5′-exonuclease activity and its absence results in rapid degradation of the mRNA (e.g., see Mol. Biol. Med. 5: 1-14, 1988; Cell 32: 681-694, 1983). Since the primary transcripts of many eukaryotic cellular genes and eukaryotic viral genes require processing to remove intervening sequences (introns) within the coding regions of these transcripts, the benefit of the cap also extends to stabilization of such pre-mRNA. This was demonstrated using mutants of capping enzymes in the budding yeast Saccharomyces cerevisiae. For example, it was shown that the presence of a cap on pre-mRNA enhanced in vivo splicing of pre-mRNA in yeast, but was not required for splicing, either in vivo or using in vitro yeast splicing systems (Fresco, L D and Buratowski, S, RNA 2: 584-596, 1996; Schwer, B et al., Nucleic Acids Res. 26: 2050-2057, 1998; Schwer, B and Shuman, S, RNA 2: 574-583, 1996). The enhancement of splicing was primarily due to the increased stability of the pre-mRNA since, in the absence of a cap, the pre-mRNA was rapidly degraded by 5′ exoribonuclease (Schwer, B, Nucleic Acids Res. 26: 2050-2057, 1998). Thus, it is also beneficial that transcripts synthesized for in vitro RNA splicing experiments are capped.
In vitro, capped RNAs have been reported to be translated more efficiently than uncapped transcripts in a variety of in vitro translation systems, such as rabbit reticulocyte lysate or wheat germ translation systems (e.g., see Paterson and Rosenberg, Nature 279: 692, 1979). This effect is also believed to be due in part to protection of the RNA from exoribonucleases present in the in vitro translation system, as well as other factors. Therefore the importance of the cap can vary with the particular translation system and its method of preparation. In any case, the use of capped transcripts can be beneficial in many cases.
The synthesis of capped RNA transcripts in vitro provides considerable value and importance for a variety of functions and applications, such as for in vitro and in vivo protein synthesis. In addition to being capped, most eukaryotic cellular and viral mRNAs have poly(A) tails on their 3′ termini. There appears to be a synergy between the 3′ poly(A) tail and the 5′-cap in increasing mRNA stability and translation. Without being bound by theory, this synergy is believed to involve an interaction between the poly(A) binding protein and the N-terminal part of the eIF4G cap binding protein, leading to mRNA circularization via a complex between the cap, the cap binding protein, the poly(A) binding protein, and the poly(A) tail. Some aspects and applications of this synergy are presented and discussed by Mockey, M et al. (Biochem. Biophys. Res. Comm. 340: 1062, 2006).
While capped mRNA remains in the cytoplasm after being exported from the nucleus, some other RNAs, such as some snRNAs have caps that are further methylated and then imported back into the nucleus, where they are involved in splicing of pre-mRNA (Mattaj, Cell 46: 905-911, 1986; Hamm et al., Cell 62: 569-577, 1990; Fischer, et al., J. Cell Biol. 113: 705-714, 1991). Transcripts with trimethylated caps have been shown to be translated with higher efficiency using Ascaris lumbicoides extracts in vitro (Maroney et al., RNA 1: 714-723, 1995).
In vivo, capping of a 5′-triphosphorylated primary mRNA transcript occurs via several enzymatic steps (e.g., see Martin, S A et al., J. Biol. Chem. 250: 9322, 1975; Myette, J R and Niles, E G, J. Biol. Chem. 271: 11936, 1996; M A Higman, et al., J. Biol. Chem. 267: 16430, 1992).
The following enzymatic reactions are involved in capping of eukaryotic mRNA:
(1) RNA triphosphatase cleaves the 5′-triphosphate of mRNA to a diphosphate,pppN1(p)Nx—OH(3′)→ppN1(pN)x—OH(3′)+Pi; and then
(2) RNA guanyltransferase catalyzes joining of GTP to the 5′-diphosphate of the most 5′ nucleotide (N1) of the mRNA,ppN1(pN)x—OH(3′)+GTP→G(5′)ppp(5′)N1(pN)x—OH(3′)+PPi; and finally,
(3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) as a co-factor, catalyzes methylation of the 7-nitrogen of guanine in the cap nucleotide,G(5′)ppp(5′)N1(pN)x—OH(3′)+AdoMet→m7G(5′)ppp(5′)N1(pN)x—OH(3′)+AdoHyc.
RNA that results from the action of the RNA triphosphatase and the RNA guanyltransferase enzymatic activities, as well as RNA that is additionally methylated by the guanine-7-methyltransferase enzymatic activity, is referred to as “5′ capped RNA” or “capped RNA”, and the combination of one or more polypeptides having the enzymatic activities that result in “capped RNA” are referred to as “capping enzyme systems” or, more simply, as “capping enzymes” herein. Capping enzyme systems, including cloned forms of such enzymes, have been identified and purified from many sources and are well known in the art (e.g., see Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001; Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 50: 101-129, 1995; and Banerjee, A K, Microbiol. Rev. 44: 175, 1980). The capped RNA that results from the addition of the cap nucleotide to the 5′-end of primary RNA by a capping enzyme system has been referred to as capped RNA having a “cap 0 structure” (e.g., see Higman, M A et al., J. Biol. Chem. 269: 14974-14981, 1994; Myette, J R and Niles, E G, J. Biol. Chem. 271: 11936-11944, 1996). Capping enzyme systems have been used to synthesize capped RNA having a cap 0 structure in vitro (e.g., see Shuman, S et al., J. Biol. Chem. 255: 11588, 1980; Wang, S P et al., Proc. Natl. Acad. Sci. USA 94: 9573, 1997; Higman M. A. et al., J. Biol. Chem. 267: 16430, 1992; Higman M. A. et al., J. Biol. Chem. 269: 14974, 1994; Myette, J. R. and Niles, E. G., J. Biol. Chem. 271: 11936, 1996; and references therein).
Capped RNA having a cap 0 structure can be further transformed in vivo or in vitro to a “cap I” structure by the action of an enzyme with mRNA (nucleoside-2′-O—) methyltransferase activity (e.g., see Higman, M A et al., J. Biol. Chem. 269: 14974-14981, 1994; Myette, J R and Niles, E G, J. Biol. Chem. 271: 11936-11944, 1996). A capped RNA with a “cap I” structure, in addition to having a 7-methyl-G cap nucleotide as the 5′ ultimate cap nucleotide, also has a 2′-O-methyl group on the 5′-penultimate nucleotide. For example, vaccinia mRNA (nucleoside-2′-O) methyltransferase can catalyze methylation of the 2′-hydroxyl group of the 5′-penultimate nucleotide of 5′-capped RNA having a cap 0 structure, as follows:m7G(5′)ppp(5′)N1(pN)x—OH(3′)+AdoMet→m7G(5′)ppp(5′)m2′-ON1(pN)x—OH(3′)+AdoHyc.
Dimethylated capped RNAs having a cap I structure have been reported to be translated more efficiently than 7-methylguanosine-capped RNAs having a cap 0 structure (e.g., see Kuge, H et al., Nucleic Acids Res. 26: 3208, 1998).
Most commonly, the RNA that has been used for in vitro capping reactions has been obtained using a T7, T3 or SP6 RNA polymerase for in vitro transcription of a template that is downstream of the respective RNA polymerase promoter, but primary RNA from other sources can also be used.
During the 1970s and early 1980s, capping enzymes were used as reagents for capping of RNAs. However, in the mid-1980s, this method for synthesis of capped transcripts was supplanted by the use of a dinucleotide cap analog to prime in vitro transcription with phage RNA polymerases (Melton, D et al., Nucleic Acids Res. 12: 7035, 1984). Since that time, post-transcriptional capping of RNA in an in vitro reaction using a capping enzyme system has not been widely used except by laboratories studying capping enzymes, and the most frequently used in vitro method to make capped RNAs having a cap 0 structure has been transcription of a DNA template with either a bacterial RNA polymerase or a bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a “dinucleotide cap analog”, also referred to as a “cap analog.” A cap analog, such as m7G(5′)ppp(5′)G (also referred to as “m7GpppG”), is a dinucleotide consisting of an outer cap nucleoside, such as 7-methyl-guanosine (m7G), and the nucleotide corresponding to the first nucleotide of the primary transcript (e.g., in this case, G). This cap analog is often used because the primary nucleotide (i.e., the most 5′ nucleotide) of most, but not all, primary RNA transcripts synthesized using phage RNA polymerase transcription systems is guanosine ribonucleotide.
Most commonly, capped RNAs are synthesized using this method by cell-free transcription of DNA templates (e.g., Contreras, R. et al., Nucl. Acids Res. 10: 6353, 1982; Yisraeli J. et al., Meth. Enzymol. 180: 42-50, 1989; and Melton, D. et al., Nucl. Acids Res. 12: 7035-7056, 1984). When capping is carried out using a cap analog in such an in vitro transcription reaction, the RNA polymerase initiates transcription by extension of the 3′-OH of the cap analog, rather than by extension of the 3′-OH of an initiating nucleoside triphosphate. Thus, if the m7GpppG cap analog is used, the initial product is expected to be m7GpppGpN. The alternative, GTP-initiated product pppGpN is suppressed by setting the ratio of m7GpppG to GTP between about 4-to-1 to about 10-to-1 in the transcription reaction mixture.
However, when using a cap analog in an in vitro transcription reaction to make capped RNA, Pasquinelli, A. et al. (RNA 1: 957-967, 1995) found that, in addition to obtaining the expected m7GpppGpN product, approximately one-third to one-half of the capped RNA products made with this m7GpppG cap analog actually had the “reverse cap” Gpppm7GpN, demonstrating that bacteriophage RNA polymerases can also use the 3′-OH of the 7-methylguanosine moiety of m7GpppG to initiate transcription. Such reverse-capped RNA molecules behaved abnormally. For example, Pasquinelli et al. reported that when reverse-capped pre-U1 RNA transcripts were injected into Xenopus laevis nuclei, they were exported more slowly than natural transcripts. Similarly, cytoplasmic reverse-capped U1 RNAs in the cytoplasm were not properly imported into the nucleus. Because the resulting capped RNAs contain about one-third to one-half reverse caps, the overall translational activity of such in vitro-synthesized mRNA is reduced and other functional properties of the mRNA may also be affected. Thus, translation of in vitro-synthesized mRNAs having such reverse caps is impaired.
To address the problem of dinucleotide cap analogs being incorporated in the reverse orientation during in vitro transcription reactions, Stepinski et al. (Nucleosides and Nucleotides 14: 717-721, 1995) and Peng et al. (Organic Letters 4: 161-164, 2002) synthesized dinucleotide cap analogs which could only be incorporated in the correct orientation because the 3′-OH of the cap nucleotide was eliminated or blocked by substitution. Since they could not be incorporated in the reverse orientation, Stepinski et al. referred to these dinucleotide cap analogs as “anti-reverse cap analogs” or “ARCAs”. Using RNA transcripts made in vitro in the presence of several different ARCAs, including m27,3′-OGpppG, it has been demonstrated that ARCA-capped RNAs result in higher translational efficiencies than RNA transcripts made in the presence of the standard m7GpppG cap analog, both for RNA transcripts translated in a rabbit reticulocyte lysate in vitro (Stepinski et al., Nucleosides and Nucleotides 14: 717-721, 1995; U.S. Patent Application No. 200301945759; Jemielity et al., RNA 9: 1108-1122, 2003; Grudzien et al., RNA 10: 1479-1487; 2004) and for RNA transcripts electroporated into mouse mammary epithelial (MM3MG) cells and translated in vivo (Grudzien et al., J. Biol. Chem. 281: 1857-1867, 2006). Mockey et al. (Biochem. Biophys. Res. Comm. 340: 1062-1068, 2006) also found that lipofection of mouse dendritic cells with a luciferase mRNA having a 3′-poly(A) tail of defined length was translated with higher efficiency if the mRNA used was capped using the m27,3′-OGpppG ARCA than if it was capped using the standard m7GpppG cap analog. The dinucleotide cap analog m27,2′-OGpppG is also incorporated only in the correct orientation and is therefore an ARCA (Jemielity et al., RNA 9: 1108-1122, 2003; Grudzien et al., RNA 10: 1479-1487; 2004). RNA capped with m27,2′-OGpppG was also translated in vitro with higher efficiency than the standard m7GpppG cap analog (Jemielity et al., RNA 9: 1108-1122, 2003; Grudzien et al., RNA 10: 1479-1487; 2004). Thus, mRNA having a cap nucleotide that is methylated in the 2′ or 3′-position was beneficial for improving translational efficiency of mRNA in vitro and in vivo.
However, although RNA can be capped by in vitro transcription of a DNA template in the presence of an ARCA, this approach has several drawbacks. First, the chemical syntheses of ARCAs (e.g., see Jemielity, J et al., RNA: 1108, 2003) are difficult (˜6 synthetic steps), time-consuming (˜12 weeks) and expensive. Also, once the ARCA is obtained, the in vitro transcription reaction is wasteful and inefficient. Due to the limiting amount of GTP in the reaction (since 80% or more of the GTP is typically substituted by ARCA), the RNA yield of the in vitro transcription with a cap analog is at best 33% of the RNA yield obtained without cap analog. Not only is the RNA yield lower using a cap analog, but also <80% of the RNA obtained is capped. Still further, the fact that the cap analog can never be incorporated to 100% limits the purity of the capped RNA product, necessitating more work to purify the product and increasing the risk that the capped RNA product will still be contaminated with impurities, including unincorporated cap analog. This is particularly detrimental if the capped RNA is to be used for medical applications, such as for therapeutics, or for clinical research, since the contaminants may produce undesired effects. Thus, there is a need for compositions and methods that provide consistent 5′ capping of RNA in a correct orientation and that increase incorporation efficiency, such as in order to improve the stability of in vitro-generated RNA transcripts (e.g., thereby increasing translational efficiency).
Also, little was known about the possibility of using a modified cap nucleotide as a substrate for a capping enzyme system to make RNA with improved properties. Published work related to use of a modified nucleotide as a substrate for a capping enzyme discouraged this approach. For example, the data of Shuman et al. (J. Biol. Chem. 255: 11588, 1980) indicated that only GTP was a good substrate for the RNA guanyltransferase activity of the vaccinia capping enzyme system; UTP, CTP, ATP, ITP, GDP, GMP and N7-methyl-GTP could not be used in place of GTP in an in vitro capping reaction. They found that 2′-dGTP seemed to have slight activity in a [32P]-PPi exchange assay in the presence of permeabilized vaccinia virions, indicating that it might be a substrate for capping, but it was only approximately 6% as active as GTP in this assay. Thus, 2′-dGTP appeared to be incorporated into capped RNA, but inefficiently. Bougie, I and Bisaillon, M (J. Biol. Chem. 279: 22124, 2004) found that the intracellular triphosphate metabolite of the antiviral nucleoside ribavirin, a nucleoside with a monocyclic base analog, was of ribavirin, was a substrate for viral capping enzyme; the ribavirin-capped RNA was more stable than uncapped RNA, but was not a functional mimic of the N7-methyl-guanosine cap with respect to translation (Yan, Y et al., RNA 11: 1238-1244, 2005; Westman, B et al., RNA 11: 1505-1513, 2005).
It would be highly desirable if there was an easier, faster, less expensive, higher yield way to make capped RNA transcripts, including modified nucleotide-capped RNA transcripts, particularly transcripts that are of a higher purity, for a variety of applications, including medical applications.
Still further, in vitro transcription in the presence of an ARCA results in RNA having a cap 0 structure. However, a capped RNA with a cap I structure could not be synthesized using an m7Gpppm2′-OG cap analog (Pasquinelli, RNA 1: 957, 1995). Also, ARCA-capped RNA has not been used as a substrate for methylation of the 5′-penultimate nucleotide of the capped RNA using mRNA (nucleoside-2′-O—) methyltransferase to obtain capped RNA having a cap I structure, which is unfortunate because 2′-O-methylation has been shown to significantly enhance translation compared to capped RNA having a type 0 structure. Therefore, it would also be highly desirable if there was a way to make modified-nucleotide-capped RNA having a cap I structure for a variety of applications, including medical applications.